Dose and location controlled drug/gene/particle delivery to individual cells by nanoelectroporation

ABSTRACT

A simple and low cost method of producing sealed arrays of laterally ordered nanochannels interconnected to microchannels of tunable size, over large surface areas, is disclosed. The method incorporates DNA combing and subsequent imprinting. Associated micro and macroscale inlets and outlets can be formed in the same process or manufactured later in low cost, non-cleanroom techniques. The techniques embrace two procedures, generating DNA nanostrands and translating these strands into nanoscale constructs via imprinting. Devices incorporating the novel arrays have a first microchannel, a second microchannel and a nanochannel that is substantially linear and which defines an axis. The nanochannel is connected at its open ends to the microchannels, which are aligned along the axis. Methods for precise dose delivery of agents into cells employing the devices in nanoelectroporation methods are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 13/177,514, filed on 6Jul. 2011 and now abandoned, which claims the benefit of U.S. 61/361,845filed on Jul. 6, 2010, now expired. Each of the prior applications isincorporated by reference as if recited fully herein.

STATEMENT REGARDING FEDERALLY-SPONSORED R & D

This invention was made with government support under Grant No.EEC-0425626 awarded by the National Science Foundation. The governmenthas certain rights in the invention.

TECHNICAL FIELD

The present invention is in the field of molecular delivery into cellsand more particularly in the field of nano-scale transfection.

BACKGROUND

The ability to deliver precise amounts of biomolecules andnanofabricated probes into living cells offers tremendous opportunitiesfor biological studies and therapeutic applications. It may also play akey role in the non-viral generation of engineered stem cells and inducepluripotent stem cells with high efficiency and non-carcinogenicproperties.

A variety of cell transfection techniques have been developed including:viral vectors, chemical methods (e.g. complexes with lipids, calciumphosphate, polycations or basic proteins) and physical methods (e.g.,particle bombardment, micro-injection and electroporation). Except formicro-injection these techniques are based on bulk stochastic processeswhere cells are transfected randomly by a large number of genes(>10⁸/cell). A disadvantage of such methods is that the injected dosecannot be controlled.

Classical chemical transfection methods including lipoplex and polyplexbased nanoparticles are often inefficient, and the level of cytotoxicityof many chemicals used is still unclear. In comparison, physicalapproaches are capable of delivering genes safely and efficientlybecause these methods can directly transfer naked genes into cells.Among them, biolistic transfection (i.e. hand-held gene gun) can beapplied to a wide variety of cell/tissue types, but it causessignificant physical damages to cells, and gold/tungsten particlecarriers may have a negative impact on cell functions. Micro-injectionis the most accurate and precise existing tool which has been widelyused to generate transgenic animal models for biomedical research.Pronuclear micro-injection, in particular, is to inject a piece ofmanipulated DNA (also called “transgene”) into one of the pronuclei ofmouse donor zygote. The injected zygotes are then transferred intorecipient surrogate mice that carry the manipulated embryos to terms.The advantage of such technique is that the gene of interest is directlyand precisely delivered into mammalian cells or specific tissues via aleast complicated procedure. Nevertheless, it requires specializedequipment, highly skilled practitioner; the quantity of injected cellsis limited within a fixed time, and most unfortunately the efficiency islow. For instance, only about 1% of injected embryos develop intotransgenic mice (10-20% of pups born), while transgenic livestock is inthe range of 5-10% offspring born. On the other hand, electroporation(EP) is the most widely used method to physically transfect cellsbecause of its technical simplicity, fast delivery, and almost nolimitation for cell types and the size of delivery materials. It hasbeen used as a research tool for investigating the biological functionsof various potential therapeutic materials in stem cells and in cancercells. In addition to in vitro study, electroporation is also used as aclinical tool for delivering anticancer drugs (e.g., bleomycin andcisplatin) for cancer therapy and DNA, RNA or DNA vaccines for genetherapy and DNA vaccination.

However, the transport of material is relatively nonspecific resultingin low cell viability and non-uniform transfection.

Bulk electroporation (BEP) is the most widely used physical method totransfect cells because of its technical simplicity, fast delivery, andalmost no limitation on cell types and size. Recently,microfluidics-based electroporation (MEP) has emerged as a newtechnology developed by a number of researchers to transfect individualcells. In MEP, a cell is located next to a small aperture (dimensions ofa few micrometers) that focuses the porating electric field to acorresponding area on the cell membrane. MEP offers several importantadvantages over BEP including lower poration voltages, bettertransfection efficiency, and a sharp reduction in cell mortality.However, the MEP delivery mechanism is similar to that in BEP: it isdiffusion dominated, electric field strengths for the two processes aresimilar, and for large transfection agents such as nucleic acids orquantum dots, entry into the cytosol is (likely) effected through anattachment onto the outside of the cell membrane followed by anendocytosis-like process. Precise dose control has not been demonstratedusing MEP.

None of the aforementioned methods has the ability to deliver a preciseamount of therapeutic/detection agents to multiple cells with a widerange of cell size.

One-dimensional nanostructures such as nanochannels (and nanotubes) arecharacterized by extremely small transverse size and resultant highdegree of spatial confinement that endow them a unique set ofproperties. When patterned laterally, these nanostructures are widelyused as critical transport devices for a variety of applications such assensing, nanomanipulation, and information processing. While numerousfabrication techniques have been developed, few can generate large andhighly ordered arrays of both nanochannels and nanowires with no defectsand low-cost. The most notable high-resolution lithographic techniquesinclude electron beam lithography (EBL) and focused ion beam milling(FIB), but they are associated with either low throughput or high-cost.Another lithographic technique, nanoimprint lithography (NIL), is ofhigh throughput and relatively low-cost, but it requires use of highlyspecialized equipment and molds prepared typically by EBL. Manyinexpensive techniques have been developed, but they are inadequate interms of high precision, low defect rate, or large area fabrication ofboth nanochannels/tubes and nanowires/strands. Moreover, thesenanostructures need to be connected to the micro/macro scale structures,such as reservoirs and channels, to form functional devices. This is nota trivial task and the lack of a low-cost solution to this problemsignificantly limits the applicability of many available nanoconstructs.

SUMMARY

This and other unmet needs of the prior art are met by compounds andmethods as described in more detail below.

Disclosed embodiments describe a new technology, nanochannelelectroporation (NEP) and demonstrate dose control—˜10%—to individualcells for a variety of transfection agents including oligonucleic acids,molecular beacons, plasmids, and quantum dots because of precise cellplacement. NEP produces an intense poration over an extremely small areaon the cell membrane: transfection agents are electrophoretically driventhrough the nanochannel, the cell membrane, and directly into thecytosol. Cell mortality from NEP is virtually zero. Small sized anddelicate cells can be transfected with precise control over dosage andtiming. The NEP device is potentially applicable for high-throughputapplications.

In an aspect of the disclosed embodiments, a micro-patterned PDMS stampwas placed on a small drop of low-cost DNA solution on a glass slide,followed by peeling off the stamp. As a result, DNA solution de-wettedthe stamp surface, leaving stretched DNA on top of the microstructuresand forming DNA nanostrands in the direction of de-wetting. For theformation of a nanochannel array, the DNA nanostrands on ridges weresputter-coated with gold. Next, the gold-coated nanostrands on the PDMSridges were placed on a solid substrate. A low-viscosity resin, e.g.ethyleneglycol dimethacrylate (EGDMA), was used to fill the spacebetween the ridges by capillary flow and then was solidified byUV-induced polymerization. The PDMS stamp was then removed, leaving thegold coated nanostrands embedded in the matrix of the cured polymer. Atthe same time, microchannels were formed from the molding of themicroridges. The cured polymer tightly surrounds the nanostrand. Toremove the gold-coated nanostrands and consequently form thenanochannels, the sample was soaked in a gold etchant.

Generally, disclosed NEP devices consist of two microchannels connectedby a nanochannel. The cell to be transfected is positioned in onemicrochannel against the nanochannel and the other microchannel isfilled with the agent to be delivered. This uniquemicrochannel-nanochannel-microchannel design allows precision placementof individual cells. A voltage pulse(s) lasting milliseconds (ms) isdelivered between the two microchannels causing transfection. Inembodiments, dose control is achieved by adjusting the duration andnumber of pulses. Alternatively, the voltage level and/or the agentconcentration can be changed.

Disclosed embodiments describe a method for the high-precisiontransfection of cells comprising providing a nanochannel electroporationdevice, the device comprising a first microchannel, a secondmicrochannel and a nanochannel having a first opening at the firstmicrochannel and a second opening at the second microchannel connectingand extending between the first and second microchannels; providing anagent to be transfected in the first microchannel; providing a cell tobe transfected in the second microchannel; and applying a voltage acrossthe nanochannel.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

A better understanding of the exemplary embodiments of the inventionwill be had when reference is made to the accompanying drawings, whereinidentical parts are identified with identical reference numerals, andwherein:

FIGS. 1 a to 1 e illustrate the steps of an exemplary method for theproduction of a nanochannel array;

FIG. 2 shows SEM images of macromolecules arranged betweenmicrochannels;

FIG. 3 is a SEM image of a nanochannel array and a schematic of a NEPchip;

FIG. 4 is a schematic of an integrated NEP and optical tweezers system;

FIG. 5 is fluorescent images of PI dye transport;

FIG. 6 is a series of graphs of fluorescent intensity profiles in cells;

FIG. 7 show fluorescent images of a single cell inside a microchannelhold near the tip of a nanochannel and a graph of changes of normalizedintensity at different NEP settings;

FIG. 8 shows data for dosage control studies using Jurkat cellstransfected with Cy3-ODN using a single pulse;

FIG. 9 shows a molecular beacon, a Jurket cell transfected with MBsproducing fluorescence and a graph of relative intensities;

FIG. 10 shows data from studies where K562 cells were treated withdifferent doses of siRNA(Mcl-1);

FIG. 11 shows the results when a cy3-labeled GFP plasmid (3.5 kbp, 0.05μg/μl) was used as a large reporter gene to visualize and detect thegene transfection process in the NEP device;

FIG. 12 shows data from transfection of Jurkat cells by a 3.5 kbCy3-labelled GFP plasmid gene in a) a representative cell taken from abulk EP run, b) NEP c) NEP+QDs;

FIG. 13 shows (a) The phase contrast image, (b) the green fluorescenceimage, (c) the combined image of a transfected cell by Q-dots, and (d)the Z stacked confocal microscope image from bottom to top of thetransfected cell with 0.2 J . . . Im step size;

FIG. 14 shows BEP transfection of pCAG-EGFP (7 kbp) conjugated with CY3inside MEF. NEP transfection of MEF cells by larger plasmid pCAG-GFP-CY3at 3 pulses of 230 V with pulse durations of 5 ms and 0.1 s separation;

FIG. 15 is a schematic of centrifugation based high throughput NEP: (a)single array chip design; (b) multi-array chip design and cell loadingprocess;

FIG. 16 is a (a) fluorescence image showing A549 cells loaded into 300microchannels after applying 100 g centrifugal force for 2 minutes; (b)design of centrifuge chip prototype used in cell loading;

FIG. 17 is a schematic of the cell loading procedure on multicell NEPchip with trapping spots;

FIG. 18 is a schematic of a NEP device;

FIG. 19 is a graph of transmembrane potentials for differentcell-nanochannel gaps; and

FIG. 20 is a graph showing transport of transfection agents into a cell.

DETAILED DESCRIPTION

The ability to deliver precise amount of biomolecules and nanofabricatedprobes into living cells with realtime imaging can offer uniqueopportunities for gene therapy, drug delivery and intercellularmolecular detection.

Disclosed embodiments describe a new technology, nanochannelelectroporation (NEP) and demonstrate dose control—˜10%—to individualcells for a variety of transfection agents including oligonucleic acids,molecular beacons, plasmids, and quantum dots because of precise cellplacement. NEP produces an intense poration over an extremely small areaon the cell membrane: transfection agents are electrophoretically driventhrough the nanochannel, the cell membrane, and directly into thecytosol. Cell mortality from NEP is virtually zero. Small sized anddelicate cells can be transfected with precise control over dosage andtiming. The NEP device is applicable for high-throughput applications.

Disclosed embodiments describe a simple and low-cost DNA combing andimprinting (DCI) method capable of simultaneously forming sealed arraysof laterally ordered nanochannels interconnected to microchannels withcontrollable sizes and rounded shape over arbitrarily large surfaceareas. Associated micro- and macroscale inlets/outlets can also beformed in the same fabrication process or added later using low-cost,non-cleanroom methods. This polymer based nanoscale transport system hasgreat potential for many challenging biomedical applications.

Disclosed methods for producing NEP devices generally embrace twoprocedures, generating macromolecular nanostrands and translating thesestrands into nanoscale constructs via imprinting. The macromolecularnanostrands are generated via a pre-patterned stamp placed in a solutionof the macromolecule. The macromolecules align with the pre-patternedmicro-ridges along the surface of the stamp. The stamp is then removedfrom the solution by first lifting one side and then the other. Thepeeling action, when performed along the axis defined by themacromolecule's alignment serves to remove one end of the macromoleculefrom the solution before the other. As the macromolecule is de-wettedfrom the solution it is stretched between two ridges. After de-wetting,the macromolecule-stamp construct is coated.

Once coated the stamp is used to imprint a nanochannel array. The newconstruct is covered in a polymer resin. The resin is cured and thestamp is removed. Due to the small size of the macromolecule, it isdetached from the coated stamp when the stamp is removed, leaving acoated macromolecule stretching between adjacent apertures defined bythe micro-ridges patterned on the stamp. The gold coated macromoleculeis the removed by a process which leaves the newly formed polymerintact. The nanochannels are thus defined on each end by largermicrochannels.

In an exemplary use of the system, the two inlets/outlets microchannelswere partially sealed by a soft-sticky PDMS film. During experiments, 40μl of a suspension containing cells in a commercial phosphate bufferedsolution (PBS) was loaded onto one side of the micro channel arraymounted on a stage of an inverted microscope with several arms formicro-motion manipulation. Over the other side of the array, 40 μl ofthe PBS containing either a fluorescent dye propidium iodide (PI), anODN, a plasmid GFP, or quantum dots (Q-dots) was loaded as modelreagents for cell transfection. Two custom electrode contacts wereinstalled on each side to generate a variety of DC pulse sequences andelectric fields by using a Bio-RAD (Gene Pulser Xcell™) power supply.The microscope was employed both for visualization and to provide thetight focus required for the laser tweezers beam. About 1 W of laserpower at 1064 nm was used to isolate and maneuver a single cellexemplary NEP.

FIG. 1 is a step-wise illustration of the process for producing amicro-/nanochannel array by DCI. FIG. 1 a shows a patterned polymerstamp 100 being removed from a solution 200. The stamp includes a seriesof micro-ridges 110 arranged along a length of the stamp. In the figure,macromolecules 120 are stretched between two adjacent micro-ridges asthe stamp is removed from the solution. In an embodiment, the stretchingof the macromolecule is aided by the angular removal/peeling of thestamp from the solution. In an embodiment, a first end of the stamp islifted creating tension along the macromolecule, stretching it as themacromolecule is removed from the solution. During the lifting of thestamp, the macromolecule is removed from the solution, and is de-wetted.In an embodiment, when the stamp is peeled from the solution, thepeeling creates stretching of the macromolecule in the direction of thede-wetting. In an exemplary embodiment, the macromolecule may be astrand of DNA.

FIG. 1 b shows an aspect of the method for producing nanochannel arrays.In an embodiment, the stamp, after having been removed from the solutionis coated by a first material. In an embodiment the stamp andmacromolecule are coated with gold. The coating may be performed by anyknown method. In an embodiment, the stamp and macromolecule are coatedwith gold via sputter coating.

FIG. 1 c shows an aspect of the method for producing nanochannel arrays.In 1 c the newly coated stamp and macromolecule are coated with a resin300. In an embodiment, the resin is a low-viscosity resin and/or a UVcurable resin. In an embodiment, the resin is ethyleneglycoldimethacrylate (EGDMA).

In FIG. 1 d the stamp 100 is removed from the resin. In an embodiment,the coated macromolecule is broken from the coated micro-ridges 140 uponremoving the stamp from the cured resin 300.

In FIG. 1 e, the coated macromolecule is removed from the nanoarray. Inan embodiment, the macromolecule is removed by chemical means includingetching and dissolving of the coated macromolecule to give amicro-/nanochannel array 150.

Example Fabrication of NEP Device

Polydimethylsiloxane (PDMS) stamps with micro-ridge designs wereprepatterned with micro-ridges. Master molds for preparing the PDMSstamps were prepared using standard soft lithography by casting PDMS(Sylgard® 184. Dow Corning) prepolymer and curing agent at 10:1 weightratio on the master. To form DNA nanostrands across the micro-ridges, a30 μl drop of 0.5 wt % DNA (75 kbp calf thymus, USB Corp) solution in TEbuffer was placed on a glass slide. The PDMS stamp was then gentlyplaced face down on the DNA solution and immediately peeled off bypulling one end of the stamp up to produce directional stretching duringthe de-wetting process.

The stamp was then sputter coated with gold (Emitech K550X, Energy BeamSciences Inc. CT, USA). The gold coated DNA nanostrands were coated with1-octane thiol by vapor deposition to facilitate de-molding of thegold-coated stamp from the cured polymer. Specifically, stamps with thegoldcoated DNA nanostrands were placed in a 10 cm-diameter Petri dishnext to 5 ml 1-octane thiol. A lid was placed on the dish and 2 h wereallowed for the vapor deposition of the I-octane thiol onto thegold-coated stamps.

Glass slides treated with Piranha were soaked in 2 wt %3-trimethoxysilylpropyl methacrylate in toluene for 12 h to graftmethacrylate groups on the slide surface. A stamp with micro-ridges andgold coated DNA nanostrands was placed face down on the treated glassslide. After ˜2 ml of EGDMA resin (99 wt % EGDMA, 1 wt % Irgacure 651initiator) was dropped on the slide near the stamp, the slide was placedin a vacuum desiccator. Once vacuum was established, the system wastilted to allow the EGDMA resin to flow towards the stamp. When theresin contacted the stamp, it immediately flowed into the space betweenthe stamp and the slide by capillary forces. Vacuum was used in thisprocess to avoid trapped air bubbles. The slide was transferred from thevacuum chamber to a small nitrogen chamber. The resin was cured using UVlight (wavelength: 365 nm, intensity: 4 mW/cm2) for 20 min undernitrogen. The adhesion between the cured polymer and the glass surfacewas enhanced by chemical bonds between surface-grafted methacrylategroups and EGDMA. The stamp was then peeled off from the slide leavingbehind the array of EGDMA micro channels connected by gold coated DNAnanostrands. To remove gold-coated nanostrands in the nanochannels, theslide was soaked in gold etchant (GE8111 Transene Company Inc., Danvers,Mass., USA) for 24 h and then thoroughly rinsed with de-ionized waterleaving behind embedded nanochannels connecting the microchannels.

Example Fabrication of NEP Device 2

A 0.5 wt % Calf thymus DNA (75 kb, USB Co,) in TE buffer was prepared toproduce an array of stretched DNA nanostrands on the microridgepatterned PDMS stamp. The stamp was then sputter coated with gold(Emitech K550X, Energy Beam Science Inc.). The stamp withmicro-patterned and gold-coated DNA nanostrands was then placed facedown on a silanized glass substrate with 3-trimethoxysilylpropylmethacrylate (Sigma-Aldrich). An ethylene glycol dimethacrylate (EGDMA),(Sigma-Aldrich) resin (99 wt % EGDMA and 1 wt % lrgacure 651 initiator)was used as an imprinting resin. The resin was cured using UV light(wavelength: 365 nm, intensity: 4 mw/cm2) for 15 min under nitrogen. Thestamp was then peeled off from the slide leaving behind the array ofEGDMA microchannels connected by gold-coated DNA nanostrands. To removegold-coated DNA nanostrands to form nanochannels, the slide was soakedin gold etchant (GE8111, Transene Company Inc.) for 48 hrs and thenthoroughly rinsed with de-ionized (DI) water leaving behind embeddednanochannels connecting the microchannels. The chip was soaked inPiranha solution (H₂SO₄/H₂O₂ 7:3) for 3 hrs to make it hydrophilic.Next, the chip was rinsed with DI water for three times and soaked in0.1% bovine serum albumin (BSA) containing DI water for 30 min to reducethe adhesion between loaded cells and substrate.

FIGS. 2 a and b are SEM images of stretched DNA nanostrands between twolong micro-ridges. Arrows depict the macromolecules. FIG. 2 a shows anembodiment with 6 μm ends. FIG. 2 b shows an embodiment with 20 μmcircular ends. These images show that the macromolecule nanostrands areoriented in the direction of de-wetting when performed in parallel to alattice axis.

In a typical single cell BEP/MEP/NEP experiment, cells (˜10⁵ cells/mL)in suspension were centrifuged for 5 min at 5000 RPM to remove theculture medium and re-suspended in phosphate buffered saline (PBS) mediafor 5-10 min prior to poration. Cell manipulation was accomplished usingan optical tweezers system built from a 3 W, 1064 nm laser (CrystalLasers) and an inverted microscope (Olympus IX-71 and Nikon EclipseTE2000). Typically, 600 mW of power was used to maneuver and hold a cellclose to the tip of the nanochannel. An electron multiplying CCD camera(Photometrics Cascade II: 512 EMCCD) detected the fluorescence emission.An electronic pulser from a Bio-RAD Gene Pulser Xcell™ electroporationsystem was used to provide the required voltage pulse sequences.Palladium wire (0.25 mm diameter, Invitrogen/molecular probes, Eugene,Oreg.) electrodes were connected to the electronic pulser for NEPstudies.

The unique microchannel-nanochannel-microchannel array design is idealfor such requirement. First, the microchannel and its curved end ensurethat each cell is confined in a well defined geometry and placed at thesame location against the nanochannel tip by force exerted from theoptical tweezers or other hydrodynamic or mechanical means. Furthermore,the very small nanochannel tip causes minimal cell deformation when thecell is pushed against the tip.

SEM Imaging: SEM images of all samples except the nanowell/nanochannelarrays were taken in a Hitachi S-4300 SEM. The high resolution SEMimages of the nanochannel were taken by a FEI Nova nanoSEM 400 equipped.

Various cell lines such as Jurkat, K562 and mouse embryonic stem cellswere used and cultured in this study. Cells were maintained in T-Flask(size: 25 cm²) at 37 DC with 5% CO₂ and subcultured and suspended in PBSbuffer for EP process.

Jurkat cells (Human T cell lymphoblast-like cell line) and K562 humanerythroleukemia cells were obtained from American Type CultureCollection (ATCC), (Manassas, Va.). Mouse embryonic fibroblasts (MEFs)were derived from 129/SV-E mice (Charles River LaboratoriesInternational, Inc MA, USA). Chronic lymphocytic leukemia (CLL) patientcells were provided by Dr. John Byrd's lab in the Comprehensive CancerCenter at The Ohio State University. The cells were routinely culturedin 25 T flask containing 5 mL of RPMI-1640 culture medium (Invitrogen,Grand Island, N.Y., USA) supplemented with 10% fetal bovine serum (FBS,Invitrogen 16000), 100 U/mL penicillin/100 μg/mL streptomycin(Invitrogen). The cells were seeded into T flasks at a concentration of3×10⁵ viable cells/mL, incubated at 37° C. in a humidified atmospherecontaining 5% CO₂, and subcultured every two days.

Once fabricated, the two rows of microchannels leads to a reservoir intowhich cells and the transfection material may be loaded, respectively.In an exemplary embodiment, an electrode was placed in each reservoir tocarry out transfection with voltage pulses between 150 and 300 voltsdepending on the need. An optical tweezers system was connected to theNEP construct allowing manipulation of a selected cell inside amicrochannel and positioning it at the tip of the nanochannel.

An example of one of the resulting nanochannels is shown in FIG. 3 withan internal diameter around 100 nm. Disclosed embodiments of the DCImethods can produce well-defined nanochannel and micro channel arraysover an area larger than several square centimeters without any defects.Lateral nanochannels alone do not constitute a functional device.Therefore, concurrent formation of the micro channels offers significantbenefits for device development. Since channel size is determined by themacromolecule size, disclosed embodiments are able to generate sealedcircular nanochannels with controllable internal diameter. Sincemacromolecules including naked DNA nanostrands below 5 nm in thicknesshave been prepared by this method and polymers have been used to imprintsub-nanometer structures by the molecular imprinting technique, DCI canthus generate nanochannel arrays with a range of channel diameters from<10 to >100 nm size. The EGDMA based nanochannel/microchannel array ishydrophobic. Therefore, organic solvents and molecules can be loaded fornanoscale fluid transport and chemical reactions. For aqueous solutionsthe array can be soaked in Piranha solution (7:3 v/v of conc. H₂S0₄ and30% H₂0₂) to make it hydrophilic, making the system convenient forsample loading in biomedical applications. The entire or a portion ofthe array surface can be covered by a soft PDMS film. This nano-featuredconstruct can be integrated with other technologies such asmicro/nano-electrodes array and magnetic or optical tweezers on anoptical/fluorescent microscope to form a nano-chip platform withreal-time monitoring and visualization capability. FIG. 3 also shows aschematic presentation of a NEP device capable of transfecting manyindividual cells with precise control of type and dosage of genes.

FIG. 4 is a block diagram of a system to isolate and maneuver individualcells. In an embodiment, the system includes a NEP device 150, a powersupply 151, a micromanipulation stage 152 a microscope 153 a COD 154 alamp 155 and a trapping laser 156. An optical tweezers system isconnected to the NEP construct allowing manipulation of a single cellinside a single micro-channel and finally holding it at the tip of thenanochannel.

Example Electropermeabilization by PI Dye: Bulk vs. NEP

Propidium Iodide (PI) dye was used to demonstrate the concept of genedelivery by nanoelectraoporation. PI dye is a membrane impermeant dyewith positive charge and low auto fluorescence. When this dye binds tothe nucleic acids (DNA and RNA) in the cell, it will produce strongfluorescence (red wavelength). FIG. 5 a-d is a series of picturessummarizing the uptake and time evolution of the PI in the single cellbulk EP and NEP experiments. In the case of bulk EP FIG. 5 c, thegradual transfer of the PI dye (total time around 18 s) was observedafter a single pulse electric bias of 700V fcm with 25 ms pulseduration, suggesting that diffusion is the dominant mechanism for PItransfer after poration. On the other hand, significant acceleration ofdye transfer was observed in the case of NEP in FIG. 5 d. At 0.03 s, astrong fluorescent signal is observed in the middle of the cell andafter just 2.3 s the entire cell turned bright by intra-cellular PI dyediffusion. This observation suggests that a localized and focusedeclectic field at the nanoscale can induce strong convective dyeinjection, resulting in the fast up-taking of the PI dye afternano-poration.

FIGS. 6 a-c show the results when the fluorescent intensity profiles intwo cells that were measured after bulk EP and NEP. FIG. 6 a shows thegradual increase of the fluorescent intensity was observed in bulk EP.On the other hand, in FIG. 6 b a sharp peak of the fluorescent intensitywas observed in the middle of the cell at t=0.03 ms (t=0 s is the onsetof EP) in NEP, suggesting that the PI dye has been transferred into thecell by the localized high electric field near the nanochannel tip. Theshape of the fluorescent intensity profile was changed from the sharppeak to smoother distribution after t>0.1 s because of internaldiffusion of the PI dye inside the cell. The whole process of the PI dyeup-taking in both bulk EP and NEP is summarized in FIG. 6 c. Calceinacetoxymethyl ester (CaAM) was used to determine cell viability afterNEP. This dye is initially non-fluorescent and cell permeable. When itdiffuses across the cell membrane, the enzyme (esterase) inside the livecell will break down and convert it to the strongly green fluorescentreagent. After NEP using the PI dye, the porated cell was incubated for30 min to fully recover cell membrane. Then, 40-50 μl of CaAM (2 μM) wasintroduced to the cell side. The porated cell inside the micro channelexhibited green fluorescent 30 min after applying the CaAM-containingsolution. The cell viability was further confirmed by using the trypanblue dye after NEP. The trypan blue dye is unable to cross an intactlive cell membrane. The porated cell by NEP did not up-take any trypanblue dye when exposed to this dye. Therefore, it was concluded that thecell is viable after the NEP treatment.

Furthermore transfection was performed with NEP, BEP and MEP of K562cells using PI. For BEP, a single 10 ms pulse with electric fieldstrength of 70 V/mm was used. A gradual increase of PI fluorescence(time ˜150 s) was observed, indicating that diffusion after poration isthe dominant mechanism for PI transfer. Similar time evolution wasobserved for electroporation by an MEP device. In contrast, significantacceleration of dye was observed in NEP during poration. The highvelocity gained by molecules in the nanochannel allow them to bedelivered into the cell during poration by a highly localized andfocused electric field inducing strong electrophoresis. See Table, fromleft to right, NEP, MEP and BEP. This unique feature allows precisedelivery of charged reagents into small cells.

Diameter 90 nm 1 um 5 um Length 3 um 5 um 5 um Voltage 180 V 150 V 60 VMax (E) (V/m) 5.80 × 10⁷ 1.60 × 10⁷ 1.10 × 10⁶ Drift Velocity 0.44 m/s0.056 m/s 0.0011 m/s Traveling Time 5.52 us 32 us 630 us

Example ODN (20 bp) Transfection with Controlled Dosage by NEP

FIG. 7 is a series of pictures and a graph showing the results offluorescent images of a single cell in a microchannel. A negativelycharged and FITC conjugated ODN (G3139, 20 bp) was delivered by NEP.FIG. 7 a shows that in the absence of any electric filed, the leukemiapatient cell was not visible in the NEP device. ODN transfer into thecell was carried out by applying one pulse of 300 V/2 mm with 25 msduration in NEP, FIG. 7 b. When the pulse length was increased from 25to 50 ms in one pulse of 300V/2 mm, the enhancement of fluorescentintensity inside the cell was observed FIG. 7 c. Finally, five pulses of25 ms at the same electric field (300 V/2 mm) were applied and the cellup-took much more ODN, resulting in a sharp increase of the fluorescentintensity FIG. 7 d. The effect of different EP settings on ODN up-takingis summarized in FIG. 7 e. These results demonstrate that embodiments ofthe NEP-device can control the dosage of small nucleic acids byadjusting the pulse duration and number of pulse. This precise controlcan offer opportunities to better understand the biological processesduring/after gene delivery.

Example ODN Transfection by NEP

To further show dosage control of NEP, Jurkat cells (˜15 μm diameter)were transfected with an 18-mer oligodeoxynucleotide (ODN, G3139)conjugated with Cy3 to allow fluorescent detection. FIG. 8 summarizesthe results of this example. NEP using a single 220 V/2 mm pulse ofvarying durations was carried out and the fluorescence signal from theODN uptake was observed, measured and is summarized in FIG. 8 a. Theamount of ODN transfected is a monotonic function of the pulse durationand near-linear from 5 to 20 ms. Similar dose vs. pulse lengthdependence was also seen for Leukemia patient cells as small as 8 μmdiameter. To demonstrate the reliability and repeatability of NEP, fivecells were transfected either simultaneously or individually, FIG. 8 b.The amount of ODN delivered showed cell to cell variation of about 10%or ±12% respectively.

Example GAPDH Transfection

The delivery of a GAPDH molecular beacon (MB) by NEP was examined.GAPDH-MB is a mRNA probe having a fluorophore at one end and a quencherat the other end of a stem-hairpin structure FIG. 9 a. Afterhybridization with complementary targets, the fluorescence is restoredby separating the fluorophore and the quencher, allowing real-timeexpression and localization of specific mRNA inside living cells. FIG. 9b shows that successful delivery of GAPDH-MB resulted in redfluorescence inside a cell. A mismatch-MB probe (scrambled) was alsotransferred as negative control to confirm the selectivity of the MB.FIG. 9 c summarizes the fluorescent intensity inside the cells atdifferent conditions. FIG. 9 c shows the relative fluorescenceintensities measured under various conditions as 220 V/2 mm within 45min after NEP. The plateau of GAPDH-MB signal at a long pulse lengths ormultiple pulses indicates that all GAPDH in the cell were detected.Since NEP can deliver probes quickly into the cell, time-dependentfouling of MB is avoided.

Example siRNA Transfection

The dosage effect on transfecting K562 cancer cells with siRNA(Mcl-1)was studied. The anti-apoptotic protein Mcl-1 promotes the survival oflymphocytes and hematopoietic stem cells and is linked to drugresistance and poor treatment outcomes in many tumor types. FIG. 10shows NEP experiments that were conducted to determine the criticaldosage of siRNA(Mcl-1) to kill K562 cells. When the pulse length islarger than 5 ms at 220 V/2 mm, a fatal dose of siRNA(Mcl-1) wasdelivered to cells (indicated by the red fluorescence). However, mostcells remained viable when the pulse length was less than 2 ms (greenfluorescence). Statistical variations of the critical dosage forindividual cells were studied using NEP with the pulse length varyingfrom 2 to 10 ms. 7-11 cells were tested for each pulse length. FIG. 10summarizes the percentages of viable and dead cells. A parallel set oftransfections using the same pulsing parameters but a scrambled siRNA isshown in the “control” columns. None of these resulted in cell death. InFIG. 10 differential interference contrast (DIC) microscopy images areshown in the left column. The green live/red dead fluorescence signalswere from a viability/cytotoxicity assay 18 hr after transfection withtwo probes that measure intracellular esterase activity and plasmamembrane integrity. For NEP pulse durations of 5 ms or longer, theinjected siRNA down-regulated the Mcl-1 protein sufficiently to induceapoptosis in K562 cells. On the right, all cells transfected with ascrambled siRNA sequence remained alive. The lower right shows cellviability under different pulse durations of siRNA(Mcl-1) injection at220 V/2 mm (Pulse length (PL)=1 ms, number of cells (N)=10; PL=2 ms,N=9; PL=3 ms, N=7; PL=4 ms, N=7; PL=5 ms, N=8, PL=10 ms, N=11).

Example GFP Plasmid (3.5 kbp) Transfection by NEP

FIG. 11 a-f shows the results when a cy3-labeled GFP plasmid (3.5 kbp,0.05 μg/μl) was used as a large reporter gene to visualize and detectthe gene transfection process in the NEP device. A single Jurkat cellwas isolated and held close to the nanochannel tip pre-filled with thecy3-labeled plasmid DNA. Then, three pulses of 250 V/2 mm with 10 msduration were applied to transfer the plasmid DNA into the cell. BeforeEP, there is no evidence of any fluorescence at the cell membrane FIG.11 a. When three electric pulses were applied, some labeled-plasmidappeared at the cell membrane by the localized electro-permeablization.With increasing time to 40 s, more labeled plasmid molecules wereinternalized into the cytoplasm. The fluorescent intensity remainedconstant in the cell after 40 s but gradually diffused inside thecytoplasm in 120-270 s. In comparison, a smaller number of the plasmidDNA molecules were observed at the cathode side of the cell in the caseof single cell bulk EP experiments under the same EP settings. There waslittle changes 600 s after EP. It has been reported that at least 30 minwas required to transfer plasmid-DNA inside the cytoplasm by endocytosisin bulk EP. Clearly, disclosed NEP methods are more efficient to deliverlarge genes into cells. After plasmid transfection, the cell culturemedium was added to the micro channels of the NEP chip. Then, the chipwas placed in a small Petri dish and incubated at 37° C. with 5% CO₂.The GFP was detected in the entire cell 24 h after poration. In acontrol experiment, the cells and GFP plasmid were mixed in anothernano-chip device and incubated for 24 h without any EP process. Therewas no green fluorescence in the control experiment. FIG. 11 a shows thetime series of cy3-labeled GFP plasmid (3.5 kbp) up-taking from 0 to 270s, after NEP, in contrast to bulk EP at 600 s, FIG. 11 b is the phasecontrast image of the incubated cell at 24 h after NEP, FIG. 11 c is thegreen fluorescence image and FIG. 11 d the blue fluorescence image ofcell nucleus stained with DRAQ-5 of the same cell, FIG. 11 e the phasecontrast image and FIG. 11 f the green fluorescence image of incubatedcells in the control experiment at 24 h.

Example Comparison Transfection

To study NEP for larger transfection agents, a Cy3-labeled GFP plasmid(3.5 kb) was used to visualize the gene transfection process of Jurkatcells. Again, we contrast conventional BEP with NEP. In BEP, DNA“complexes” formed on the outside of the cell followed by anendocytosis-like passage into the cytoplasm that takes nearly an hour,FIG. 12 a. This is similar to previously reported observations.Migration to the nucleus and subsequent transcription required manyhours. GFP fluorescence was not observed even 12 hrs after transfection.On the other hand, NEP injects the plasmid directly into the cytoplasm:within 40 s of poration, a significant Cy3 fluorescence was observedinside the cell membrane. Migration of the DNA to the nucleus andtranscription occurred under 6 hrs FIG. 12 b. For higher genetransfection, nanoparticles such as gold or quantum dots (QDs) can beused to facilitate delivery, roughly analogous to the role of the needlein micro-injection. FIG. 12 c shows the NEP transfection of a Jurkatcell using a mixture of QDs conjugated with the COOH group with theaforementioned GFP plasmid. This procedure led to strong GFP expressionwithin 3 hrs. FIG. 12 c also shows that QDs were delivered uniformlyinside the Jurkat cell using NEP. A viability/cytotoxicity assay,confirmed the transfected cells shown in the Figure to be alive.Injecting a uniform spatial distribution of nanoparticles into a cell isvaluable for many scientific studies, but not achievable by BEP, MEP orother methods. In FIG. 12 Cy3 fluorescence is shown in the black andwhite photos taken shortly after transfection. In the color photos, theCy3 fluorescence shows as yellow, the nucleus (DRAQ-5 fluorescence) isblue, and the GFP signal is green. On the left, it took nearly an hourfor GFP to pass the cell membrane in bulk EP. GFP fluorescene was seenafter 18 hrs. In the center, NEP injected DNA directly into thecytoplasm. GFP fluorescence was seen in 6 hrs. On the right, a mixtureof plasmid and quantum dots facilitated delivery of DNA into cellnucleus. A strong GFP signal was seen within 3 hrs. QDs were delivereduniformly inside the cell. Bulk EP was carried out on NEON™ transfetcionsystem at 1325 V with 10 ms pulse length and 3 pulses. Two pulses of 260V/2 mm with 5 ms pulse length and 0.1 separation were used in NEP.

Example Q-Dots Up-Taking by NEP

To explore the delivery of nanoparticles by NEP, Q-dots (InvitrogenCorp.) conjugated with the COOH group (8.0 nM) were introduced in thenano-chip device and transfected to a Jurkat cell by NEP using a singlepulse of 250 V/2 mm with 25 ms duration. FIG. 13 summarizes the resultsfrom the Q-dots up-take by NEP to Jurkat cell. Many Q-dots can betracked inside the transfected cell as shown in FIG. 13 a-d. FIG. 13 ashows the phase contrast image, (b) the green fluorescence image, and(c) the combined image of a transfected cell by Q-dots, (d) the Zstacked confocal microscope image from bottom to top of the transfectedcell with 0.2 μm step size.

Example Plasmid Transfection

To investigate the capabilities of an NEP platform for transecting cellswith large plasmids, such as cell reprogramming factors the NEP deliveryof pCAG-GFP plasmids (˜7 kbp) into mouse embryonic fibroblast (MEF)cells as a model system was examined. FIG. 14 compares the delivery ofpCAG-EGFP by BEP (Neon™ transfection system, one pulse at 1300 V, pulseduration=30 ms) and NEP (three pulses at 220 V, pulse duration=5 ms,separation between pulses=0.1 s). In BEP, Cy3-plasmids were delivered byan endocytosis-like passage into the cytoplasm that took several hours(FIG. 14, left). After 12 hours of BEP transfection, a weak GFPfluorescence signal was detected₁₃. On the other hand, NEP transferredlarge plasmids directly into the cytoplasm (FIG. 14, right) by NEP.Strong green fluorescence expression of pCAG-GGFP was detected insideMEF after 7 hours of NEP transfection.

To handle large numbers of cells, a high throughput process is neededfor NEP. Centrifugation has been widely used in the biomedical field forcell and biomolecule separation.

FIGS. 15 a-b show two exemplary designs. In contrast to the opticaltweezers based NEP chip, four reservoirs are used in this design, twofor the transfection agent (e.g. DNA, molecular probe or nanoparticles)and two for the cell. The length of the cell-side microchannels isgreatly shortened to allow only one cell per microchannel. The chip issealed similar to the optical tweezers device. The cell loading andde-loading process includes seven steps: 1) Load biomolecules using thesame method as used for optical tweezers devices and load cells to oneof the cell reservoirs using a pipette. 2) Place the chip near theperiphery of a disk that is mounted on a rotation device. (3) Move thecells from the cell reservoir to the main connection channel by usingthe centrifugal force at a low rotation speed. Excess cells will flow tothe other cell reservoir. The biomolecule side needs to be completelysealed to prevent any material loss. (4) Demount the chip rotate it 90degrees and remount it to the disk in order to alter the direction ofthe force so as to push the cells into the microchannels. Move the cellsto the end of the cell-side microchannels by using the centrifugal forceat a higher rotation speed. This action also pushes cells against thetip of the nanochannels to ensure good cell-channel contact. Conductelectroporation. (6) Add Trypsin to the cell side to release the cellsfrom the microchannel surface. (7) Turn the chip around on the disksurface and use the centrifugal force to de-load cells into the originalcell reservoir.

FIG. 16 shows a result in which ˜200 cells are transfected within a fewminutes. Although several microchannels did not receive any cells andsome channels got more than one cell, the electrical behavior of eachmicrochannel will be the same with or without cell(s) because the majorelectric resistance (and thus the voltage drop) is across thenanochannel. This simple and fast approach demonstrates the feasibilityof a high throughput NEP. Alternatively, for example, a cell trappingspot can be deposited in front of each microchannel in the mainconnection channel as shown in FIG. 17.

In an embodiment, this cell trapping spot can be tethered antibodymolecules to capture exactly one cell per microchannel during the cellloading stage. Excess cells can be washed away from the main connectionchannel. By using trypsin to release the cells from the trapping spots,each cell can move into the microchannel opposite it using centrifugalforce.

The current microchannel-nanochannel-microchannel NEP array shown inFIG. 15 can hold 200-300 cells and it can be extended to 1,000 cells. Byadding multiple arrays on a single chip and placing a number of chips onone rotation disk, a large cell population (i.e. from 10,000 to >50,000)can be porated uniformly and simultaneously using NEP. Although theaforementioned high throughput microchannel-nanochannel-microchannel NEParray′ chip still cannot compete with the conventional bulkelectroporation methods in regard to cell numbers (10⁴ vs. 10⁶) andassay time (minutes vs. seconds), the unique features of high precisiondosage control and fast and high efficacy transfection (i.e. bypassingendocytosis and endosome escape) provided by NEP may often more thanoffset these limitations.

Example Modeling

As a representative example, NEP transfection of a 15 μm diameter cellby COOH conjugated quantum dots with a total diameter 20 nm was modeled.A single 10 ms, 200 V pulse is used in the model. FIG. 18 shows aschematic of the experimental layout. The nanochannel is a 90 nmdiameter and 3 μm long channel. The conductivity of the PBS solutionused for the experimental work is 1.5 S/m. This is reduced to 0.8 S/m bythe addition of the low conductivity solution containing the quantumdots. The microchannels are about 40 μm diameter and 1000 μm long. Thusthe resistance is: R_(nc)=1/G_(nc)=(σ_(buffer)A/I)⁻¹=590 MΩ. Themicrochannels are about 40 um diameter and 1000 um long giving aresistance R_(MC)=1 MΩ. This is negligible compared to the nanochanneland relevant cell membrane resistances. Thus, the microchannel ismodeled as wires, treat the fluids in the microchannels as equipotentialsurfaces and note that variations in the placement of the electrodeshave negligible impact on the process. The cell interior will be treatedas an equipotential surface. The cell membrane is divided into two partsas shown in FIG. 18. Membrane 1 refers to the section adjacent to thenanochannel and membrane 2 is the rest of the cell membrane. Transverseconduction within the cell membrane between the two sections is ignored.Electrically an intact cell membrane is modeled as a resistance inparallel with a capacitance. Membrane surface capacitances are morereliably known: 1×10⁻² F/m⁻². For the present conditions, the resistanceand capacitance of membrane 1, adjacent to the nanochannel are 8×10¹³Ωand 6×10⁻¹⁷ F respectively. Membrane 2 has resistance and capacitance of710 MΩ and 7 pF respectively. Putting all of these together produces theelectrical model of the system for an intact cell. Note: the appliedvoltage satisfies: V_(applied)=V_(NC)+V_(TM1)+V_(TM2). This model isvalid so long as all of the membranes remain intact. In visualizing theNEP process the exact value of this minimum threshold doesn't mattervery much—somewhere between 0.2 V and 5 V.

In this model the NEP process is readily described. Upon application ofa voltage pulse, almost instantly all of the voltage appears acrossmembrane 1, facing the nanochannel. That membrane reaches the criticalvoltage in nanoseconds and porates. Assuming that the now-poratedmembrane 1 represents a very low resistance, the second “outer” membranestarts charging in a predictable way:

V _(TM2) =V _(applied) R ₂/(R ₂ +R _(NC))(1−e ^(−t/(RNPC2)))≈0.54V_(applied)(t/4.1)

where t is measured in milliseconds. This predicts that for a highvoltage pulse (100 V or greater), the second membrane charges to acritical voltage in 10's to 100's of microseconds and must, in thismodel porates. Again, assuming that the 2_(nd) porated membrane has anegligible voltage drop across it, the “poration current” flowingthrough the cell is limited by the nanochannel: I≈V_(applied)/R_(NC)

Electrophoresis dominates the transport of the transfection agentsthrough the nanochannel. This is a feature that is novel to NEP becauseof the high fields used. Since almost all of the voltage drop is acrossthe nanochannel, the field in the channel is very nearly pulse voltagedivided by the channel length. For these conditions, this is about 70MV/m. We use for the mobility of the quantum dot (QD), μ_(QD)=−1×10⁻⁸m²V⁻¹ s⁻¹ which produces a drift velocity for the QD of about 700 μm/ms.Thus transfection transport occurs as follows. QDs enter the nanochannelthrough a combination of drift and diffusion and are rapidly, within afew microseconds, swept to the cell. In the numerical simulations it isshown that fringe fields extend into the cell and can “inject” QDsthrough the cell membrane and into the cytoplasm.

The fields generated during NEP are truly enormous—several tens of MV/m.Experimentally, a striking feature of the data is that, in contrast towhat is observed in other types of EP, for NEP large transfectionagents—DNA and QDs—appear almost instantly inside the cytoplasm. Thissuggests that large pores are created in the cell membrane if not thecomplete breakdown/disappearance of the membrane opposite thenanochannel during the electrical pulse.

It is clear that pores will form in the cell membrane near thenanochannel tip at high enough voltages. Those pores allow the passingof biomolecules into the cell attached to the nanochannel duringporation.

To further analyze the NEP process and to compare NEP and MEP, finiteelement simulations of the electrostatic problem were carried out. Theanalysis is based on the setup for cell EP depicted in FIG. 18. Theproblem is simplified to be axial symmetrical. From the top view, thechannel for EP with diameter d=90 nm and length l=3 μm is embeddedbetween two larger microchannels with width W=40 μm. A cell withdiameter D=15 μm is placed at the end of left microchannel. An electricpotential, Vp=200 V, is applied across the whole device of total lengthL=2 mm. The table below includes values for additional model parameters.The electrostatic problem was solved to study the cell's transmembranepotential (TMP), material dynamics in the channel during EP, and theelectric field at various points within the apparatus and the cellitself.

Symbol Value Definition D 15 μm Cell diameter d_(m) 5 nm Cell membranethickness σ_(e) 0.8 S/m Electrical Cond. of external medium σ_(i) 0.2S/m Electrical cond. Of cytoplasm σ_(m) 5 × 10⁻¹ S/m Electrical cond. Ofcell membrane

For a cell with no net surface charge, the static electric field isgoverned by the Laplace equation:

∇×(σ∇V)=0

where V is the electric potential and σ is the electric conductivity.Once V is solved, the electric field E could be determined by E=−∇V andTMP V_(m) can be obtained from V_(m)=V(S_(ext))−V(S_(int)) where S isthe surface of cell membrane and “ext” and “int” denote the external andinternal surfaces. To circumvent the difficulty in the mesh generationfor the thin cell membrane, the concept of contact resistance in heatconduction is adopted here. The computer simulation is carried out usingcommercial FEM software, COMSOL Multiphysics™.

To study the sensitivity of cell placement in NEP, a gap between thecell edge (θ=0) and the outlet of the nanochannel is included in thesimulation. Gap sizes of 10, 5, and 2 nm were studied. Simulations wereperformed both for systems with intact cells and for systems containinga pore at the interface with the nanochannel. A single pore was createdon the cell whose area was that defined by the contact surface with the90 nm diameter cylinder of the nanochannel. In the simulation, the poreon the cell is a section of the membrane with higher electricconductivity, 0.2 S/m. FIG. 19 shows the magnitude of TMPs of the cellwithout and with the pore with an enlarged view at the low voltage inthe inserted. If the cell is intact (solid lines), the max(TMP)increases from 20 to 55 V as the gap is decreased from 10 to 2 nm.Therefore, before the cell is electroporated, the peak of TMP is indeedsensitive to the cell position. However for all values, the cellmembrane would certainly porate; nanopore(s) would be formed withinmicroseconds once the TMP is over a critical value. As a result, thesharply peaked distribution of TMP lasts an extremely short timecompared to the total electroporation pulse length (severalmilliseconds). After the cell is electroporated, the peak TMP will dropdramatically and the TMP for the area away from the defect will increase(solid lines to dashed lines). The sharply peaked TMP distributionchanges to being near-uniform except for the area of the pore itself.After the cell is electroporated by the nanochannel the sensitivity tothe gap is reduced. The exact position of the cell is unimportant in thesense that for all 3 positions, a 200 V pulse should be more than enoughto cause electroporation.

The transport of transfection agents was also modeled. To do this thecomputed electric field was taken and numerically integrated theequation of motion for a molecule traveling down the center of thenanochannel. It was assumed that the transfection agent passed unimpededthrough the cell membrane. Within the cell itself the quantum dotmobility was reduced by a factor of 60. This is consistent with measuredreduction of the quantum dot diffusion coefficient relative to that ofwater. The simulation reproduces the observation of the circuit-basedmodel, above, that molecules are rapidly transported through the 3 μmlong nanochannel. In addition, we observe that fringing fieldselectrophoretically “inject” charged particles a significant distancepast the cell membrane and into the cytoplasm. FIG. 20 shows theresults.

Precise dose control in the absence of cell damage is nearly impossiblewith conventional techniques. However, These requirements can be easilyachieved using NEP, but not MEP for the following four reasons. (1) Thecell surface area affected by electroporation in NEP is less than 1%that in MEP, leading to much less damage to the cell. This isparticularly critical for small primary cells. (2) In NEP the “shot” isgenerated by the large electrophoretic force imposed on theto-be-delivered biomolecules both within the channel and through thecell membrane. For this purpose, a high electric voltage is necessary togenerate a large electric field and a correspondingly highelectrophoretic force. Dose control is achieved because transport oftransfection agents is dominated by electrophoresis occurring during theelectrical pulse. In comparison, voltages used in MEP result in lowelectric fields within the microchannel and cannot produce meaningfulelectrophoretic forces. Consequently, for MEP, delivery of biomoleculesinto the cell still has to rely largely on molecular diffusion andendocytosis after poration. If a higher voltage (say hundreds volts) wasused in MEP to increase molecule acceleration, the cells would notsurvive. (3) Although the electroporation duration is very short (ms),the formed pores on the cell membrane may take many seconds to re-seal.To prevent cell damage caused by ions leaking out of the cell during andafter poration and poor dosage control caused by uncontrolledbiomolecules taken up by the cell after poration, molecular diffusionthrough the channel during and after poration must be minimized. Massdiffusion through a channel is inversely proportional to the channelarea (d₂) and exponentially proportional to the inverse of the channellength squared (i.e. ˜exp(αt//₂)). A nanochannel (<100 nm) is able tolimit mass diffusion much better than a microchannel (>1 μm). For largemolecules such as nucleic acids, the Brownian motion and sterichindrance further limit the transport of molecules through the verysmall nanochannels. (4) The precision of cell placement around thechannel plays a major role on the delivered dosage. For MEP, a portionof the cell often deforms into the channel under a force (either vacuumor hydrodynamic). In some cases, particularly for smaller primary cells,the whole or a major portion of the cell is squeezed into the channel.It is difficult to keep the cell deformation and shape the same for eachmicrochannel. This is not an issue formicrochannel-nanochannel-microchannel NEP design because the cellplacement and cell shape can be kept consistent as long as the cell isin close contact with the nanochannel tip and there is virtually no celldeformation.

When K562 cells were drawn into microchannels they confirmed nicely ontothe microchannel surface in close contact with the nanochannel tip in anexemplary NEP device when loaded using optical tweezers. The cellplacement and shape remained unchanged hours after poration. Precisionof cell placement is critical for repeatable dosage controlled deliveryto individual cells. The repeatable transfection and dose control in NEPindicates that it can reliably and accurately position cells withrespect to the nanochannel tip.

The terms “a” and “an” and “the” and similar references used in thecontext of describing the disclosed embodiments (especially in thecontext of the following claims) are to be construed to cover both thesingular and the plural, unless otherwise indicated herein or clearlycontradicted by context.

Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided herein isintended merely to better illuminate the disclosed embodiments and doesnot pose a limitation on the scope of the disclosed embodiments unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element essential to the practice of thedisclosed embodiments or any variants thereof.

Groupings of alternative elements or embodiments disclosed herein arenot to be construed as limitations. Each group member may be referred toand claimed individually or in any combination with other members of thegroup or other elements found herein. It is anticipated that one or moremembers of a group may be included in, or deleted from, a group forreasons of convenience and/or patentability.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention(s).Of course, variations on the disclosed embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventors expect skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention(s)to be practiced otherwise than specifically described herein.Accordingly, this disclosure includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above describedelements in all possible variations thereof is encompassed by thedisclosed embodiments unless otherwise indicated herein or otherwiseclearly contradicted by context.

Having shown and described an embodiment of the invention, those skilledin the art will realize that many variations and modifications may bemade to affect the described invention and still be within the scope ofthe claimed invention. Additionally, many of the elements indicatedabove may be altered or replaced by different elements which willprovide the same result and fall within the spirit of the claimedinvention. It is the intention, therefore, to limit the invention onlyas indicated by the scope of the claims.

What is claimed is:
 1. A method for the production of a nanochannelelectroporation device comprising the steps of: providing a stamp withchannel-defining micro-ridges, the ridges arranged in parallel along anaxis from a first end to a second end; providing a solution of amacromolecule capable of forming strands; placing the stamp into thesolution; directionally stretching the macromolecule across two alignedmicro-ridges; removing the stamp from the solution; coating the stampand the macromolecule with a metal; applying a resin to the coatedstamp; curing the resin; and removing the stamp from the resin.
 2. Themethod of claim 1, wherein: the macromolecule provided is a strand ofDNA.
 3. The method of claim 1, further comprising the step of: treatingthe surface of the coated stamp prior to applying the resin.
 4. Themethod of claim 1, wherein: the resin applied is a low-viscosityprepolymer resin.
 5. The method of claim 1, further comprising the stepof: removing the macromolecule from the cured resin.
 6. A method for thehigh-precision transfection of cells, comprising the steps of: providinga nanochannel electroporation device, the device comprising a firstmicrochannel, a second microchannel and a nanochannel having a firstopening at the first microchannel and a second opening at the secondmicrochannel connecting and extending between the first and secondmicrochannels; providing an agent to be transfected in the firstmicrochannel; providing a cell to be transfected in the secondmicrochannel; and applying a voltage across the nanochannel.
 7. Themethod of claim 6, wherein: applying the voltage transfects the cellwith a predetermined dose of the transfecting agent.
 8. The method ofclaim 7, wherein: the voltage is applied for milliseconds to transfectthe cell with a dose error of less than 20%.
 9. The method of claim 6,wherein: the agent to be transfected is chosen from the group consistingof plasmids, biomolecules, small molecules, fluorophores, dyes, RNA,DNA, and proteins.
 10. The method of claim 6, wherein: the cell isviable after transfection.
 11. The method of claim 6, further comprisingthe step of: loading at the second opening the cell to be transfected.12. The method of claim 11, wherein: the step of loading the cell isachieved by applying a centrifugal force.
 13. The method of claim 11,wherein: the step of loading the cell is achieved by optical tweezers.14. The method of claim 6, wherein: a pulse number and duration of thevoltage applied determines an amount of the dose transfected.